Identification of Protein Kinase C Phosphorylation Sites Involved in Phorbol Ester-Induced Desensitization of the Histamine H1 Receptor

• Published in: Molecular pharmacology Vol. 55; no. 4; p. 735
• Main Authors: Fujimoto, K, Ohta, K, Kangawa, K, Kikkawa, U, Ogino, S, Fukui, H
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.04.1999
• Subjects: Amino Acid Sequence; Animals; CHO Cells; Cricetinae; Humans; Molecular Sequence Data; Mutation - drug effects; Peptide Fragments - metabolism; Phosphorylation; Protein Kinase C - metabolism; Receptors, Histamine H1 - genetics; Receptors, Histamine H1 - metabolism; Sequence Homology, Amino Acid; Serine - metabolism; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0026-895X; 1521-0111

The histamine H 1 receptor (H1R)-mediated signaling cascade is inhibited by phorbol ester-induced protein kinase C (PKC) activation. Cloning studies of the H1Rs have shown that several potential PKC phosphorylation sites are located in the third intracellular loop of H1R. To elucidate the molecular mechanism of PKC-mediated desensitization, we identified amino acid residues that are involved in the desensitization of the H1R. Two amino acid residues (Ser 396 , Ser 398 ) were determined to be PKC phosphorylation sites by in vitro phosphorylation studies using a series of synthetic peptides. Treatment with phorbol ester decreased histamine-induced accumulation of inositol phosphates in Chinese hamster ovary cells expressing the H1R with a rightward shift in the EC 50 value, which implies the uncoupling of the receptor from the G protein. Site-directed mutagenesis studies showed that substitution of alanine for Ser 398 but not for Ser 396 markedly attenuated the effect of phorbol ester, which suggests that the Ser 398 residue was primarily involved in PKC-mediated desensitization.